Colicin A is a water-soluble toxin that forms a voltage-gated channel in the cytoplasmic membrane of target bacteria. In the present thesis, we aimed at studying the closed channel state, the membrane insertion mechanism, the acidic pH induced molten globule state and the interaction of colicin A in living E. coli cells. For that, we used Electron Paramagnetic Resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) method to explore the structural details of colicin A. The EPR studies of the membrane-bound colicin A (reconstituted into proteoliposomes) suggest the transmembrane orientation of the hydrophobic hairpin in the closed channel state. The pH dependent membrane insertion studies indicate that the membrane binding efficiency is significantly enhanced at pH < 3. Moreover, in the presence of a membrane potential, the pH induced membrane-bound state is able to open channels in the liposomes. The membrane-bound conformation (induced by acidic pH) is similar to the conformation of reconstituted colicin A which support the umbrella model for the closed channel state of colicin A. The studies on pH dependent conformational changes suggest that colicin A forms a molten globule at pH 2. The molecular details of pH induced conformational changes were analyzed by molecular dynamic simulations. The results of the MD simulations agree with the EPR results. Conformational changes of colicin A upon interaction with living E. coli cells could also be followed. Comparison between colicin A in wild type (WT) cells and tolB knock-out mutants suggest that the observed conformational changes originate from colicin A which has been already translocated to the inner membrane.
| Identifer | oai:union.ndltd.org:uni-osnabrueck.de/oai:repositorium.ub.uni-osnabrueck.de:urn:nbn:de:gbv:700-2007071316 |
| Date | 12 July 2007 |
| Creators | Pulagam, V. Lakshmi Padmavathi |
| Contributors | Prof. Dr. Heinz -Jürgen Steinhoff, Prof. Dr. Hildgund Schrempf |
| Source Sets | Universität Osnabrück |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/zip, application/pdf |
| Rights | http://rightsstatements.org/vocab/InC/1.0/ |
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